Project Summary The optic nerve head (ONH) is the primary site of axon injury in glaucoma, a neurodegenerative disease that is the leading cause of irreversible blindness affecting 76 million people worldwide. Elevated intraocular pressure (IOP) is the only modifiable risk factor for glaucoma, and lowering IOP is the only available strategy to slow the progression of vision loss. Thus, there is a critical need for novel therapeutic strategies targeting the site of axon injury within the ONH. In order to develop novel ONH-targeted treatments for glaucoma, we must determine the early cellular events that lead to ONH axon injury. Astrocytes (local glia within the ONH) provide structural and metabolic support for axons. In neurodegenerative disorders including glaucoma, astrocytes become ?reactive? and display structural and molecular changes. In several glaucoma models, including ours, significant ONH astrocyte actin- and intermediate filament-based cytoskeletal reactivity occurs prior to observable axon injury. Whether IOP-dependent ONH astrocyte reactivity is neuroprotective or helping drive disease, or whether modulation of these reactive responses alters axon vulnerability to elevated IOP remain unclear. Using a rodent model of acute IOP elevation, our preliminary data demonstrate that ONH astrocytes react by retracting their actin-based cellular extensions and reducing connexin43 labeling (an astrocyte gap junction protein involved in maintaining astrocyte syncytial isopotentiality, and reliant on the actin cytoskeleton for localization). Furthermore, we show that actin cytoskeletal stabilization (using the Rho kinase inhibitor fasudil) significantly reduces ONH astrocyte cytoskeletal & gap junction reactivity and protects axons in this model. Lastly, using an in vivo surgical strategy developed in our lab, we show that ONH astrocytes can be modulated by local small molecule delivery to the ONH. In this proposal, we will determine the role of ONH astrocyte cytoskeletal reactivity in axon degeneration after acute IOP elevation, by combining our rodent model with local and systemic delivery of small molecule modulators of the cytoskeleton. Next, we will examine the mechanistic role of ONH astrocytic connexin43 in IOP-dependent axon degeneration using small molecule and genetic strategies to suppress connexin43 in our rodent models. Axon- and astrocyte-specific immunofluorescence of ONH tissue will be used to determine the extent of axon injury and astrocyte responses within the ONH at various time points after IOP elevation. In the course of this work, we will address the role of the ONH astrocyte cytoskeleton and gap junctions in IOP-dependent axon degeneration, as well as their modulation as a novel strategy for axon protection. The ultimate long-term goal of this project is to bring to light new astrocyte-specific therapeutic targets to reduce the burden of glaucomatous vision loss worldwide.